Antigen specific multi epitope-based anti-infective vaccines

ABSTRACT

The invention provides peptide vaccines comprising the signal peptide domain of selected target antigens of intracellular pathogens. The peptide vaccines of the invention contain multiple class II and class I-restricted epitopes and are recognized and presented by the majority of the vaccinated human population. The invention provides in particular anti tuberculosis vaccines. The invention further provides compositions comprising the vaccines as well as their use to treat or prevent infection.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/588,887 filed on May 8, 2017 which is a continuation of U.S. patentapplication Ser. No. 13/384,286 filed on Jan. 16, 2012, which is aNational Phase of PCT Patent Application No. PCT/IL 10/00569 havingInternational filing date of Jul. 15, 2010, which claims the benefit ofpriority of U.S. Patent Application Nos. 61/225,957 filed on Jul. 16,2009. The contents of the above applications are all incorporated byreference as if fully set forth herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to peptide vaccines directed againstintracellular pathogens with pan HLA class I and class II bindingproperties, as well as to pharmaceutical compositions containing thepeptide vaccines and methods for treating or preventing infectionscaused by intracellular pathogens.

BACKGROUND OF THE INVENTION

Intracellular pathogens are the main cause for increased morbidity andmortality worldwide. The list of intracellular infectious agents thathave had a significant impact on global health and economy includesviral pathogens such as human immunodeficiency virus (HIV), hepatitis Band C virus (HBV and HCV), Influenza, Epstein Barr virus (EBV),protozoan parasites which are the causative agents of Chagas disease,Malaria, Toxoplasma and Leishmaniasis and bacterial pathogens includingagents responsible for Tuberculosis (TB), Chlamydia and Leprosy. Inspite of decades of research, there is very little progress in thedevelopment of effective vaccines against these pathogens, most of thevaccines being the live attenuated pathogens.

Mycobacterium tuberculosis (Mtb) is one of the most ubiquitous pathogensin the world. It is estimated that roughly one third of the world'spopulation is infected, resulting in 3 million deaths per year.Tuberculosis continues to be a major public health issue in many partsof the world due to a) the relatively long period of treatment requiredto cure it, b) the emergence of drug-resistant strains and c) the risein HIV infection as a cofactor that facilitates the transmission andprogression of the disease.

Currently, a live attenuated strain of Mycobacterium bovis (BCG) is usedas a vaccine for children. However, this is not sufficiently effectiveas it has variable efficiency (0-80%), immunity tends to wane after10-15 years and it fails to control dormant or new infection.

Despite this, BCG still has value for example in its efficacy againstmeningeal TB and leprosy. Nevertheless, a more effective vaccine isessential in order to control the spread of TB more effectively. Inparticular, there is a demand for more effective preventive “preinfection” vaccines as well as “post infection” vaccines that could beadministered against a background of BCG immunization and/orpre-existing Mtb infection.

WO 2008/035350 discloses signal peptide based therapeutic vaccinecompositions targeted against various tumor associated antigens.

SUMMARY OF THE INVENTION

The present invention provides an antigen specific peptide vaccine whichis able to induce strong, comprehensive response in the majority of thepopulation against a target pathogen. More specifically, but withoutwishing to be limited to a single hypothesis, such a vaccine preferablycombines activation of both CD4⁺ and CD8⁺ T cells via multiple class IIand class I-restricted epitopes which are present within the internalsequences of the vaccine and are derived from the same antigen, and willlead to inhibition of intracellular development of the pathogen therebypreventing infection.

The present invention thus provides a peptide vaccine comprising atleast one signal peptide domain of at least one target protein of anintracellular pathogen or a pathogen-induced host protein wherein saidsignal peptide domain is recognized and presented by more than 50% ofthe MHC (major histocompatibility complex) class I and MHC Class IIalleles in the vaccinated human population.

In one aspect, the present invention relates to peptide vaccinescomprising at least one signal peptide domain of at least one targetantigen of an intracellular pathogen.

In one embodiment, the present invention relates to peptide vaccinesconsisting of at least one signal peptide domain of at least one targetantigen of an intracellular pathogen.

In one embodiment the peptide vaccines of the invention comprise atleast one signal peptide of proteins selected from the group consistingof the Tuberculosis antigens-BPBP1, Antigen 85B, Antigen 85B-Precursor,Lipoprotein lpqH, Putative lipoprotein IprB precursor, Putativelipoprotein IpqV precursor, Beta gluconase putative, Hypotheticalprotein MTO 213, Protease, ATP dependent helicase putative, Hypotheticalprotein MT1221, BCG, Hypothetical protein Rv0476/MT04941 precursor,Hypothetical protein Rv1334/MT1376 precursor, beta-Lactomase precursor;the Malaria P. Falciparum antigens-Circumsporozoit protein precursor,Malaria exported protein-1, Liver stage antigen (LSA-1), Sporozoitsurface antigen 2, MSP1, Protein Antigen; the Malaria P. Vivaxantigens-Cytoadherence linked asexual protein, Membrane protein PF12,Exported protein 2, Circumsporozoite-protein related antigen,Circumsporozoite protein precursor, Merozoite surface protein 3 alfa,CTRP adhesive protein invasive stage; The Toxoplasma gondiiantigens-GRA-1, SAG1, Surface antigen SAG1, Surface Antigen P22, Rhoptryprotein 10; The EBV antigens-Glycoprotein GP85 and BCRF-1, the CMVantigens, Glycoprotein B, RAE-1 (human), Unique short US8 glycoproteinprecursor, US9 Protein, US7; the human Prion protein; the HIVantigens-Envelope Glycoprotein, reticulocalbin-2 precursor (human),neuronalacetycholine reccep. alfa 3 (human), Envelope polyprotein GP′160precursor and Transforming membrane receptor-like protein; the Herpesvirus 8 antigen—HHV-8 glycoprotein E8-1.8, the Influenza antigens—HA andNeuraminidase and the human HCV related protein CEP and Angioprotein 1receptor precursor (Tables 1-3).

In one embodiment the signal peptide-derived peptide vaccine comprisesthe amino acid sequence selected from the group consisting of the aminoacid sequences listed in Table 1, SEQ ID NOs: 1-54.

The MHC prediction calculations were adjusted according to dominantClass I and II alleles in populations of various territories. Theoutcome is MHC-prediction coverage for three main territories: Westernpopulation (listed in table 1), Sub Saharan Africa (listed in table 2)and South West Asia (listed in table 3).

According to one specific embodiment, the present invention relates to apeptide vaccine comprising the signal peptide domain of a tuberculosisprotein.

Preferably, said peptide is not longer than 40 amino acids.

According to specific embodiments said peptide vaccine comprises atleast one amino acid sequence selected from the group consisting of:

(SEQ ID NO. 1) MKIRLHTLLAVLTAAPLLLAAAGCGS designated herein VXL-200;(SEQ ID NO. 2) MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAdesignated herein VXL-201; (SEQ ID NO. 4) MKRGLTVAVAGAAILVAGLSGCSSdesignated herein VXL-203; (SEQ ID NO. 7) MKTGTATTRRRLLAVLIALALPGAAVAdesignated herein VXL-206; (SEQ ID NO. 8)MAAMWRRRPLSSALLSFGLLLGGLPLAAPPLAGA designated herein VXL-207;(SEQ ID NO. 9) MRFAQPSALSRFSALTRDWFTSTFAAPTAAQAdesignated herein VXL-208; (SEQ ID NO. 12) MLVLLVAVLVTAVYAFVHAdesignated herein VXL-211; (SEQ ID NO. 13) MLLRKGTVYVLVIRADLVNAMVAHAdesignated herein VXL-212; (SEQ ID NO. 15) MRPSRYAPLLCAMVLALAWLSAVAGdesignated herein VXL-214;

According to further embodiments said peptide vaccine consists of atleast one amino acid sequence selected from the group consisting of: SEQID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8, SEQ IDNO. 9, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 15.

In a specific embodiment said peptide vaccine consists of at least oneamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 12, and SEQ ID NO. 13.

In one embodiment, the peptide vaccine comprises a combination of atleast two signal peptides.

In another aspect, the invention encompasses a polypeptide vaccinecomprising a recombinant polypeptide comprising at least one signalpeptide domain of a target protein of an intracellular pathogen or apathogen-induced host protein.

In a specific embodiment, the polypeptide vaccine comprises at least twosignal peptide domains of target proteins derived from an intracellularpathogen or a pathogen-induced host protein. In a further embodimentsaid at least two signal peptide domains are selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 7,SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO.15. In a further specific embodiment, the polypeptide vaccine comprisesat least two signal peptide domains selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 12, and SEQ IDNO. 13 (corresponding to the peptides VXL201, VXL 203, VXL 208, VXL 211,and VXL 212, respectively). Said polypeptide preferably comprisesadditional amino acid sequences in between the VXL peptides. Withoutwishing to be bound by theory, these additional amino acid sequences areintended to reduce the hydrophobicity of the chimeric molecule. In onespecific embodiment the ratio between the VXL peptides and theadditional amino acid sequences in the chimeric polypeptide is about1:1.

In one specific embodiment said polypeptide vaccine comprises the aminoacid sequence denoted in SEQ ID NO: 55, or an amino acid sequenceshaving at least 85% homology with SEQ ID NO: 55, or an amino acidsequence having at least 90% homology with SEQ ID NO: 55, or an aminoacid sequence having at least 95% homology with SEQ ID NO: 55.

In another specific embodiment, said chimeric polypeptide comprises morethan one copy of the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4,SEQ ID NO. 9, SEQ ID NO. 12, and SEQ ID NO. 13, namely the chimericpolypeptide comprises repetitions of the VXL peptides.

The present invention further encompasses a polynucleotide encoding thechimeric polypeptide, as well as vectors comprising same and host cellscomprising said vector or polynucleotide. Said polynucleotide mayfurther comprise at least one restriction site. Such a restriction sitemay be employed for removing one or more of the VXL peptides from thechimeric polypeptide.

The present invention also concerns use of the peptide vaccinesdescribed above in the preparation of pharmaceutical compositions fortreating or preventing pathogenic infections.

The invention further concerns pharmaceutical compositions comprising atleast one of said peptide vaccines and a pharmaceutically acceptablecarrier or diluents, and the use of said peptide vaccines or saidpharmaceutical compositions as anti-pathogenic vaccines to treat orprevent infections. In one embodiment said infections are caused bymycobacterium tuberculosis.

Optionally, said pharmaceutical compositions further comprise anadjuvant

The present invention also encompasses pharmaceutical compositionscomprising a combination of at least two peptide vaccines, therebyallowing vaccination against several different antigens of the samepathogen or against several different pathogens.

The invention further concerns nucleic acid molecules encoding saidpeptides, and antigen presenting cells (APC), e.g. dendritic cells,presenting said peptides, as well as pharmaceutical compositionscomprising said nucleic acid molecules, or said cells.

The invention also concerns use of the peptide vaccines for enrichmentof T cell populations in vitro. Thus obtaining a peptide-specificenriched T cell population.

The invention further concerns the use of said nucleic acid molecules,cells, or pharmaceutical compositions comprising same as anti-infectivevaccines to treat or prevent pathogenic infections. In one embodimentsaid infections are caused by mycobacterium tuberculosis, or malaria.

Further aspects of the present invention are directed to a method fortreating or for preventing infections by administering thepharmaceutical compositions of the present invention to a patient inneed thereof.

The pharmaceutical compositions of the invention may be adapted for usein combination with other anti pathogenic agents, for example,antibodies or antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 is a list of vaccine candidates for Tuberculosis. The amino acidsequence of the vaccine candidates is presented in the right column. Theleft and mid columns represent internal terminology (VXL) and therespective protein designation.

FIGS. 2A-2B are graphical representations of proliferation responsesinduced by VXL tuberculosis vaccine candidates (VXL) on naïve peripheralblood mononuclear cells (PBMC). Results are indicated as a positivestimulation index SI>2 (FIG. 2A) or as an average stimulation index (SI)and standard deviation (FIG. 2B). SI was calculated by dividing the CPM(counts per minute) obtained in an analyzed sample to the CPM in acontrol sample (medium). Results are an average of 8 differentexperiments using 11 donors. PHA (phetohemaglutinin), TT (tetanusToxoid) and tuberculosis Purified protein Derivative (PPD) served aspositive controls while HuFab (human Fab antibody fragment) was used asa negative control.

FIGS. 3A-3B are graphical representations of proliferation responsesinduced by VXL tuberculosis vaccine candidates on TT or PPD positivenaïve PBMC. The graphs present the % of positive >2 proliferation inresponse to the vaccine candidates (VC) in PBMC derived only from donorswhich are also positive (>2) for the TT antigen (FIG. 3A) or the TBantigen PPD (FIG. 3B). In this experiment TT or PPD were considered as100% positive. Results are an average of 5 (FIG. 3A) and 4 (FIG. 3B)different experiments.

FIGS. 4A-4B are graphical representations of proliferation responsesinduced by VXL tuberculosis vaccine candidates on naïve PBMC derivedfrom PPD-positive versus PPD-negative donors. The results are presentedas average of direct stimulation index (SI) with SD (FIG. 4A) or as % ofpositive proliferation response (SI>2) in PPD positive donors (blackbox) and PPD negative donors (white box) (FIG. 4B). Results are anaverage of 8 different experiments, using 11 donors.

FIG. 5 is a table providing characteristics of tuberculosis patientsincluding their ethnic group and disease-associated parameters.

FIGS. 6A-6B are graphical representations of proliferation responses ofPBMC obtained from patients with active Mtb in response to stimulationin vitro with various Mtb peptides of the invention (designated VXLpeptides). Results are indicated as a positive stimulation index SI>2(FIG. 6A) or as an average stimulation index (SI) and standard deviation(SD) (FIG. 6B) and represent an average of 4 different experiments using14 TB patients.

FIGS. 7A-7B4 are graphical representations of cytokine secretion, asmeasured by quantitative ELISA, by PBMC obtained from naïve donors. PBMCwere stimulated in vitro, using the tuberculosis peptides of theinvention for a period of 48 hrs. FIG. 7A represents the percentage ofdonors with positive (x>0.1 ng/ml) cytokine secretion from the 11evaluated naïve donors, upon stimulation with various peptides of theinvention. FIGS. 7B1-7B4 represent one of three similar experimentsshowing cytokine secretion (ng/ml) and proliferation response (asindicated by SI) to stimulation with the most potent VC VXL208 (FIG.7B1), VXL211 (FIG. 7B2), VXL212 (FIG. 7B3), or VXL203 (FIG. 7B4) in 5 ofthe 11 donors.

FIGS. 8A-8B4 are graphical representations of cytokine secretion, asmeasured by quantitative ELISA, by PBMC obtained from tuberculosis (Mtb)patients. PBMC were stimulated in vitro, using the tuberculosis peptidesof the invention for a period of 48 hrs. FIG. 8A represents thepercentage of donors with positive (X>0.1 ng/ml) cytokine secretion fromthe 6 evaluated donors with active TB, upon stimulation with variouspeptides of the invention. FIGS. 8B1-8B4 demonstrate cytokine secretion(ng/ml) and proliferation response (as indicated by SI) to stimulationwith VXL203 (FIG. 8B1), VXL208 (FIG. 8B2), VXL211 (FIG. 8B3), or VXL212(FIG. 8B4) in 6 tuberculosis patients.

FIGS. 9A-9B are graphical representations of proliferation responses ofPBMC obtained from naïve donors (FIG. 9A) and Mtb patients (FIG. 9B) inresponse to stimulation in vitro with various Mtb peptides of theinvention. Two types of peptides were used in the experiment: peptideshaving purity of more than 70% and peptides having purity of more than90%. The results are presented as stimulation index (SI).

FIG. 10 is a graphical representation of proliferation responses of PBMCobtained from naïve donors in response to stimulation in vitro withvarious Mtb peptides of the invention as compared with other known ornovel epitopes derived from the same tuberculosis antigens (controlpeptides). The results are presented as stimulation index (SI).

FIGS. 11A-11D are graphical representations of cytotoxic activity of Tcell lines generated against the peptides of the invention: Anti VXL 203CTL (FIG. 11A), Anti VXL 208 CTL (FIG. 11B), Anti VXL 211 CTL (FIG.11C), and Anti VXL 212 CTL (FIG. 11D). Results are presented as percentlysis of target cells, macrophages (Mp) pre-loaded with the respectiveVXL peptide. Macrophages loaded with control antigen match, or PPD, orunloaded macrophages served as controls.

FIGS. 12A-12E are graphical representations of lysis of Mtb infectedmacrophages by various VXL-peptide induced T cell lines. FIGS. 12A and12B—represent data obtained with naive donor-derived T cell lines andFIGS. 12C, 12D and 12E—represent data obtained with Mtb patients-derivedT cell lines.

FIG. 13A is a table summarizing the presence or absence of lysisactivity of various VXL-peptide specific T cell lines (Anti 201, Anti203, Anti 208, Anti 211, and Anti 212) obtained from naïve donors andMtb patients.

FIG. 13B is a table summarizing the range of activity in thecytotoxicity assay.

FIG. 14 is a table summarizing the characteristics of four VXL-peptidespecific T cell lines (Anti 201, Anti 203, Anti 208, and Anti 211). Thetable shows the percentage of CD4+ or CD8+ cells which express theeffector cell marker CD44^(high) and the effector memory markerCD62L^(high).

FIGS. 15A-15D are schematic representations of FACS analysis resultsshowing the percentage of IFN-gamma production by various peptidespecific T cell lines. FIG. 15A—represents data obtained with theVXL203-peptide specific T cell line; FIG. 15B—represents data obtainedwith the 203A-peptide specific T cell line; FIG. 15C—represents dataobtained with the VXL211-peptide specific T cell line; and FIG.15D—represents data obtained with the 211A-peptide specific T cell line.

DETAILED DESCRIPTION

The present invention is based on the use of signal peptide sequences asa source for the preparation of promiscuous T cell epitope peptidevaccines with excellent immunodominant properties against intracellularpathogens. The uniqueness of the peptide vaccines of the invention stemsfrom their ability to bind multiple MHC class I and MHC class IIalleles. This feature enables the generation of a robust immune responsevia combined simultaneous activation of both CD4+ and CD8+-restricted Tcell clones and coverage of the majority of the population via bindingto multiple class I or II alleles, while using a single, relativelyshort sequence of amino acids. As used herein the term CD4+ or CD8+refers to cluster of differentiation 4 or 8, respectively.

In addition, and without wishing to be bound by theory, the peptidevaccines of the invention have an internal adjuvant property whichelevates their immunodominant properties. Furthermore, said peptidevaccines have TAP independent pathway for entering the ER thereby, theycan better deal with the TAP-related immune escape mechanism andconsequently with the pathogen's ability to down regulate MHC molecules.Suitable antigens for vaccination in accordance with the invention(hereinafter defined as “target antigens”) include two main groups:

-   -   A. Pathogen associated antigens: These are antigens which are        known or have the potential to be immunogenic and to be        expressed by host cells (Target Cells) at a preferred site and        time during the process of infection.    -   B. Host associated antigens: These are antigens which are        unregulated and overexpressed by target cell post infection and        although considered to be self can induce specific immunity to        pathogen infected cells at a preferred site and time during the        process of infection.

The present invention provides pathogen specific vaccines which arecapable of inducing an effective T-cell response against anintracellular pathogen, and which are based on the signal peptidesequence of proteins associated with the pathogen infection.

It is well known that host defense against intracellular pathogensdepends on effective cell-mediated immunity (CMI), in which interactionsbetween T cells and macrophages are crucial. A principal effectormechanism of CMI is through the production of key type I cytokines,particularly interferon-γ, IL-2, and IL-12 which are being produced byantigen-specific activated CD4+ helper T cells, antigen specific CD8+Cytotoxic T-cells, and antigen presenting cells (APC). Such antigenspecific CD8+ Cytotoxic T-cells possess a high lytic capacity forinfected cells, and are also capable of eliminating parasites. Suchcells also have life-long memory immunity.

It is widely accepted today that protective immunity againsttuberculosis relies on the activation of T cells rather than B cells.Within the T cell family, it is the CD4+ T cells which are thought to beimportant in fending off Mtb. However, CD8+ cells are also known toparticipate in the anti-Mtb immune response, but their relativeimportance during the progressive stages of the disease remains elusive.T cells are known to exert their function at least partly throughsecretion of cytokines. In particular interferon-gamma (IFN-Gamma) andinterleukin-12 (IL-12) have been ascribed beneficial roles in protectionagainst TB and exceptional susceptibility to TB has been described inhuman individuals who are genetically deficient for IFN-Gamma receptor,the IL-12 receptor or IL-12.

In spite of a multitude of treatments against Malaria, around 2 to 3million lives are still lost every year with approximately 90% of thesein Sub-Saharan Africa.

Immunization with radiation-attenuated Plasmodium sp. sporozoiteslremains the ‘gold standard’ for malaria vaccine development; the vaccineprevents both the development of the clinical symptoms of malaria andthe transmission of the disease. Nevertheless, despite intense researchfor decades, the mechanisms and antigenic targets of protective immunityto malaria remain poorly understood.

It is believed today that an efficient protective malaria vaccine needsto elicit a combination of robust CD4+ and CD8+ against key antigensexpressed during the early stages of infection i.e. in the draininglymph node and liver. The combination of both effector cells is a key.While Helper CD4 T cells are thought to be a key in fending offPlasmodium, the action of effective Cytotoxic T cells that act viacytokine secretion and specific lysis of infected cells (probablyhepatocytes) is also critical.

Lymphotropic Herpesviruses (LH), Epstein-Barr Virus (EBV),Cytomegalovirus (CMV), and human Herpesvirus-6 (HHV-6), establish alifelong persistent infection in most people. They usually produceunapparent infection or transient immune compromise in otherwise healthyhosts but are able to cause life-threatening primary or reactivatedinfections in individuals with congenital or acquired T-cellimmunodeficiency. The spectrum of diseases caused by LH, and CMV is welldocumented in patients undergoing bone marrow transplantation (BMT) ororgan transplantation and in individuals infected with humanimmunodeficiency virus (HIV). Monitoring of EBV DNA load in thecirculation is clinically valuable for the management of EBV-associateddiseases, including primary infections in early adulthood which exhibitacute inflammatory diseases known as infectious mononucleosis andchronic forms associated with different Human malignancies such asnasopharyngeal carcinoma (NPC), Gastric carcinoma, and certain lymphomassuch as Burkitt's Lymphoma and Hodgkin's disease. CMV is ubiquitous inhumans. Around the world the mean seropositivity rate varies withlocation, race, and socioeconomic status. However in any location,almost all the individuals eventually become infected, ranging from60-70% in urban U.S. cities to 100% in Africa. The immunosuppressiveregimens used for transplanted patients predispose them to CMV disease.Infected organs or blood transfusions are the most common sources ofinfection. In these patients the severity of the disease is related tothe degree of immunosuppression. Morbidity due to donation from aseropositive donor to seronegative recipient is usually higher andmanifested as intestinal pneumonia and hepatitis.

Antigen Processing in Eukaryotic Cells

In Eukaryotic cells, protein levels are carefully regulated. Everyprotein is subject to continuous turnover and degradation. The pathwayby which endogenous antigens are degraded for presentation by class IMHC molecules utilizes the same pathways involved in the normal turnoverof intracellular proteins. Intracellular proteins are degraded intoshort peptides by a cytosolic proteolytic multifunctional system calledproteasome present in all cells. The proteasome involved in antigenprocessing includes three subunits LMP2, LMP7 and LMP 10.

The peptidase activities of Proteasome containing LMP 2, 7 and 10generates peptides with preferred binding to MHC class I. The transportof proteasome-degraded peptides into the endoplasmic reticulum (ER)involves an ATP dependent transporter designated TAP. This is amembrane-spanning heterodimer consisting of two proteins TAP1 and TAP2.TAP has a higher affinity to peptide with hydrophobic Amino acids, thepreferred anchor residues for MHC class I.

In this regard, signal peptide sequences from self or foreign antigenswhich are highly hydrophobic are more likely to get a better access toMHC molecules via their preferred binding to TAP1 and TAP2 molecules(Immunology 5^(th) addition R A Goldsby, T J Kindt, B A Osborne and JKuby).

Since peptide presentation on MHC class I is a key for mounting aneffective cellular immunity, intracellular pathogens are constantlytrying to abrogate this machinery. One mechanism for achieving this goalis via specific mutation in the TAP transporter. Practically, TAP1 orTAP2 mutations halt the MHC peptide interaction in the ER leading tolimited presentation of MHC-peptide complexes on the cell membrane. Aswill be explained in the next paragraph, SP sequences are the onlypeptides which deal with these mutations as they have a TAP-independentpathway for penetrating into the ER.

Signal Peptide Processing

In both bacteria (prokaryotic) and eukaryotic cells, proteins destinedfor secretion or for insertion into cellular membranes need to betargeted appropriately. Genes that encode such proteins specify a short,amino-terminal signal peptide (termed also signal sequence) that isrequired for the protein to find its way to the membrane. Although allsignal peptides have a common motif, there is no homology between signalpeptides in different proteins. The signal peptide is proteolyticallyremoved in the ER after its targeting role has been performed. In bothtypes of organisms, a signal recognition particle (SRP) is responsiblefor recognizing and binding to a signal peptide sequence at the aminoterminus of a growing, membrane-bound protein. The SRP then targets theribosome that is synthesizing the protein to either the endoplasmicreticulum (ER) in eukaryotes, or to the bacteria plasma membrane inbacteria. The SRP binds to ribosomes at the site of the exit tunnel andinteracts with the N-terminal end of newly synthesized protein. If theprotein contains a SP sequence then protein synthesis is temporarilyarrested. The complex is directed to the membrane surface and the end ofthe protein is inserted through a pore in the membrane. Proteinsynthesis then continues and the newly synthesized protein is insertedinto the endoplasmic reticulum in eukaryotes or across the plasmamembrane in bacteria. In the final step a SP specific peptidase isreleasing the SP into the lumen of the ER where it can bind MHCmolecules. This is a normal process which runs in parallel to theproteasome machinery. Moreover, it was well demonstrated in variousreports that SP linked to short sequences and even isolated SP per se(without the entire protein chain), can penetrate the ER via the samespecific peptidase.

The mechanisms of antigen presentation for proteins derived fromintracellular bacteria are more complicated since these proteinspresumably need to first reach the host cytosol and only than penetrateinto the host's ER for MHC presentation. There is limited informationabout this process but there is sufficient evidence for CTL activityagainst key epitopes in bacteria. Potential mechanisms are thefollowing:

-   -   1. Phagosome: Intracellular organelles which lyse bacteria and        transport the processed antigen for direct presentation on MHC.    -   2. Bacteria secreted protein (having bacteria SP sequences), can        be degraded in the cytosol by the proteasome and enter the ER        via the TAP machinery.    -   3. SP sequences from Bacteria having also the binding motif of        the human SP peptidase can, as explained above, penetrate        independently the ER. This is a proteasome independent mechanism        that can potentially also in the absent of functional TAP        transporter.

Selection of the Vaccine Candidate

The present invention provides signal peptide (SP)-based vaccinecandidates against intracellular pathogens. Such vaccines may bepreventive or therapeutic vaccines.

Such intracellular pathogens include, but are not limited to bacteria,Mtb. Antigenic targets for development of an anti-pathogen vaccine canbe selected based on any desired criteria, for example, using thefollowing key parameters:

-   -   Pathogen or host antigens which are differentially expressed in        pathogen-infected cells (Host Cells).    -   Eligible targets for an immune assault    -   Predicted as binding for class I, and II (and therefore likely        to be immunogenic) in the majority of the population; and    -   Having less than 80% homology with any self (human host)        antigen.

Preferably such targets are expressed at a preferred site and timeduring the process of infection, e.g. in the case of Mtb infection inthe human Lung and lymph nodes or in the case of Malaria infection inthe human liver or spleen.

Searching for a Comprehensive List of Eligible Target Proteins

Several information sources can be used for selection of relevant targetproteins, for example: Published scientific articles, patents or patentapplications; sequence databases such as Blast or uniprot search; asignal peptide database website which provides a direct access to thesignal sequence domain of Mammals, Drosophila, Bacteria and Viruses(http://www.signalpeptide.de/index.php?m=listspdb); and a list ofpublished epitope antigens in the website of Immune Epitope Database andAnalysis Resource (IEDB).

Checking the List for Eligible Targets for an Immune Assault

Each protein was checked for eligibility to become an immune targetaccording to the criteria defined above.

Proteins with the following attributes were removed from the list:

-   -   0. Host proteins that are sub-cellularly located in organelles        or in localization that does not require transport from the        ER-Golgi, and thus have no signal peptides (e.g. purely        cytoplasmic proteins (e.g. ATP-citrate synthase)).    -   1. Host proteins that are ubiquitously expressed in many tissues        (e.g. TUBB, RBM4).    -   2. Immune-related proteins (e.g. PSME2, CD213a2, M-CSF).    -   3. Proteins which are expressed in cells which do not have MHC        presentation (i.e. RBC).

Identifying the Signal Peptides (SP)

Proteins that were eligible targets for an immune assault were checkedfor the presence of a signal peptide. The SignalP 3.0 program was usedto determine the signal peptide sequence(http://www.cbs.dtu.dk/service/SignalP/). Information was also confirmedfor SP sequences isolated in other websites e.g. “The signal peptidedatabase”. The program uses both a neural network (NN) algorithm and aHidden Markov models (HMM) algorithm. A sequence was considered to be asignal peptide whenever a score of over 0.3 was received in one or moreof the algorithms. In the case of bacterial antigens, targets wereevaluated for the presence of bacterial SP and human SP. In addition,the human SP was required to be smaller in size from the bacterial SP.

Checking for Predicted Binding of Predicted Peptides

To check whether peptides from the 17-40 amino acid signal peptides bindto frequently appearing human leukocyte antigen (HLA) haplotypes, wecollected information on HLA allele frequency from the dbMHC sitebelonging to the ncbi.

First, the alleles of HLA class I (HLA-A, B, C), and HLA class II(HLA-DRB1) which appear most frequently in a selected population weredetermined. Online prediction programs that were used:

-   -   0. BIMAS: used for most of the predictions for HLA class I        alleles.    -   1. NetMHC: used for the prediction of certain HLA class I        alleles which are more frequent in selected populations e.g.        South West Asia and Sub Sahara Africa. This software uses for        certain alleles the Neuronal network prediction methodology and        for other alleles the Scoring Matrix methodology.    -   2. Propred: used to predict most DRB1 genotypes.    -   3. Immune Epitope: used for the prediction of the HLA-DRB1-0901        genotype that is not predicted by Propred.

Defining Differential Strength of Binding

In each of the programs used, various differential strengths of bindingwere defined:

-   -   1. BIMAS: Strong=peptide score of 100+, Medium=10-100,        Weak=5-10.    -   2. NetMHC: for neuronal network Strong=peptide score of 1-50,        Medium=50-500, Weak=500-5000 nM. For scoring matrix there is a        specific threshold score given for each allele. Nevertheless,        the threshold was always in the following range, Strong=peptide        score in the range of above 12.5-15 Weak=1-8.5, Medium=was in        the range of 8.5-(12.5-15) depending on the allele.    -   3. Propred: Strong=top 1% of binders, Medium=1-2% of binders,        Weak=2-3% of binders    -   4. Immune Epitope: Strong=IC₅₀ of 0.01 nM-10 nM, Medium=10-100        nM, Weak=100-10,000 nM    -   5. MHC2Pred: Strong=cutoff 1.0, medium=cutoff 0.5,        Weak=cutoff 0. As serotype prediction is expected to be less        accurate than genotype prediction, only high and medium binders        were predicted with MHC2Pred.        Determining the Predicted Percentage of Population that has        Alleles that have Predicted Binding Peptides within a Specific        Signal Peptide

To calculate the probability that a patient (or a population) has one ormore alleles predicted to bind a certain signal peptide, a statisticcalculation using complementary probabilities was used. Independentdistribution of alleles in the population was assumed.

Calculation: if peptide X was predicted as a peptide that binds to onlyfour HLA-class I alleles: HLA-A1 (frequency 0.1), HLA-B2 (freq=0.2),HLA-B3 (freq.=0.3), and HLA-C4 (freq. 0.4) then the probability that itwould bind neither of these alleles is the product of the probabilitiesthat it would bind neither HLA-A 1 (1-0.1), nor HLA-B2 (1-0.2), norHLA-B3 (1-0.3), nor HLA-C4 (1-0.4) therefore the probability is:

(1−0.1)(1−0.2)(1−0.3)(1−0.4)=0.3024.

The probability that the patient has one or more of the binding allelesis 1 minus the probability that he would have none of the bindingalleles:

1−0.3024=0.6976

The calculation was done separately for the HLA class I alleles, theHLA-class II alleles (genotypes), and the HLA class II alleles(serotypes). Each list contained no overlapping alleles (e.g. HLA-A02and HLA-A0201).Peptides that would bind in the majority (>50%) of the population (bothin the HLA class I and in the HLA class II alleles) were furtherfollowed.Table 1 provides a comprehensive list of selected targets suitable foraddressing the Western population with respect to Class I and Class IIallele coverage.

TABLE 1 SEQ. % % No. Protein Disease length SP sequence MHC-I MHC-II  1BPBP1 Tuberculosis 26 MKIRLHTLLA VLTAAPLLLA AAGCGS 75 52  2 Antigen 85BTuberculosis 40 MTDVSRKIRA WGRRLMIGTA AAVVLPGLVG 85 52 LAGGAATAGA  3Antigen 858-Precursor leprae 38MIDVSGKIRA WGRWLLVGAA ATLPSLISLA GGAATASA 82 36  4 Lipoprotein IpqHTuberculosis 24 MKRGLTVAVA GAAILVAGLS GCSS 86 52  5Putative lipoprotein (Possible lipoprotein) leprae 20MRHKLLAAIY AVTIMAGAAG CSGGTQA 66 45  6 Beta gluconase putativeTuberculosis 31 MLMPEMDRRR MMMMAGFGAL AAALPAPTAW A 85 41  7Hypothetical protein MT0 213 Tuberculosis 27MKTGTATTRR RLLAVLIALA LPGAAVA 83 48  8 Protease Tuberculosis 34MAAMWRRRPL SSALLSFGLL LGGLPLAAPP LAGA 81 41  9ATP dependet helicase putative Tuberculosis 32MRFAQPSALS RFSALTRDWF TSTFAAPTAA QA 84 37 10 Hypotethical protein MT1221Tuberculosis 37 MLSRTRFSMQ RQMKRVIAGA FAVWLVGWAG GFGTAIA 61 41 11 BCGTuberculosis 23 MRIKIFMLVT AVVLLCCSGV ATA 69 52 12Un char protein Rv0476/MTO4941 prec Tuberculosis 19 MLVLLVAVLVTAVYAFVHA84 50 13 Un char protein Rv1334/MT1376 prec Tuberculosis 20MLLRKGTVYVLVIRADLVNAMVAHA 79 50 14 Putative lipoprotein IprB precTuberculosis 24 MRRKVRRLTLAVSALVALFPAVAG 71 50 15Putative lipoprotein IpqV prec Tuberculosis 25 MRPSRYAPLLCAMVLALAWLSAVAG83 47 16 Beta-Lactomase precursor Tuberculosis 30MRNRGFGRRELLVAMAMLVSVTGCARHASG 81 43 17Circumsporozoit protein precursor P. Falciparum 18 MMRKLAILSV SSFLFVEA74 52 18 Malaria exported protein-1 P. Falciparum 22MKILSVFFLV LFFIIFNKES LA 86 52 19 Liver stage antigen (LSA-1)P. Falciparum 23 MKHILYISFY FILVNLLIFH ING 84 52 20Sporozoit surface antigen 2 P. Falciparum 25 MNHLGNVKYL VIVFLIFFDL FLVNG84 52 21 MSP1 (merozoit surface protein 1 precur) P. Falciparum 20MKIIFFLCSF LFFIINTQCV 82 52 22 Protein Antigen P. Falciparum 25MNIRKFIPSL ALMLIFFAFA NLVLS 85 45 23Cytoadherence linked asexual protein, P. Vivax 24MTSLRNMRVF FLFVLLFISK NVIG 79 52 CLAG 24 Membrane protein PF12 P. Vivax23 MRIAKAALCG QLLIWWLSAP AEG 78 45 25 Exported protein 2, putativeP. Vivax 21 MKVSYILSLF FFLIIYKNTT T 83 48 26Circumsporozoite-protein related antigen P. Vivax 21MKLLAAVFLL FCAILCNHAL G 75 41 27 Circumsporozoite protein precursorP. Vivax 22 MKNFILLAVS SILLVDLFPT HC 78 52 28Merozoite surface protein 3 alfa P. Vivax 23 MKHTRSVTLY LFLLTLCAYL TGA84 43 29 CTRP adhesive protein invasiv stage P. Vivax 23MNKSFLLIAS YFCLVVHLGT VIA 82 52 30 GRA-1 Toxoplasma gondii 24MVRVSAIVGA AASVFVCLSA GAYA 75 48 31 SAG1 Toxoplasma gondii 39MSVSLHHFII SSGFLTSMFP KAVRRAVTAG VFAAPTLMS SO 45 32 Surface antigen SAG1Toxoplasma gondii 30 MFPKAVRRAV TAGVFAAPTL MSFLLCGVMA 87 45 33Surface Antigen P22 Toxoplasma gondii 26 MSFSKTTSLA SLALTGLFVV FQFALA 8241 34 Rhoptry protein 10 Toxoplasma gondii 28MGRPRWPLPS MFFLSLLCVS EKRFSVSG 85 45 35 Glycoprotein GP85 EBV 17MQLLCVFCLV LLWEVGA 71 52 36 BCRF-1 EBV 23 MERRLVVTLQ CLVLLYLAPE CGG 8752 37 Glycoprotein B CMV 25 MESRIWCLVV CVNLCIVCLG AAVSS 75 52 38Retinoic acid early inducible gene-1 CMV,NH1 30MRRISLTSSP VRLLLFLLLL LIALEIMYNS 86 52 (RAE-1) 39Unique short US8 glycoprotein precursor CMV 21 MRRWLRLLVG LGCCWVTLAH A69 45 40 US9 Protein CMV Human 27 MILWSPSTCS FFWHWCLIAV SVLSSRS 63 45herpesvirus 5) 41 Unique short US7 glycoprotein precursor CMV Human 27MRIQLLLVAT LVASIVATRV EDMATFR 80 52 herpesvirus 5 (HHV5) 42 Hu PrPViroid/Hibatitis 22 MANLGCWMLV LFVATWSDLG LC 76 52 43Envelop Glycoprotein HIV 30 MRVKEKYQHL WRWGWKWGTM LLGILMICSA 81. 45 44Hu reticulocalbin-2 precursor HIV 25 MRLGPRTAAL GLLLLCAAAA GAGKA 82 4145 Hu neuronalacetycholine reccep. alfa 3 HIV 29MALAVSLPLA LSPPRLLLLL LSLLPVARA 84 52 46Envel polyprotein GP 160precursor HIV 29 MRATEIRKNY QHLWKGGTLL LGMLMICSA85 41 47 Envel Protein GP160 precursor HIV 29MRVKGIRRNY QHWWGWGTML LGLLMICSA 80 45 48Transforming membrane receptor-like HIV HHV-8 18 MLLCIVCSLL VCFPKLLS 6948 protein. 49 HHV-8 glycoprotein E8-1.8 HV8 26MSSTQIRTEI PVALLILCLC LVACHA 83 52 50 Angioprotein 1 receptor precursorHCV 22 MDSLASLVLC GVSLLLSGTV EG 71 52 51 C-Reactive protein HCV HIV 18MEKLLCFLVL TSLSHAFG 67 45 52 Hemagglutenin precursor NH1 17MKANLLVLLC ALAAADA 75 45 53 Hemagglutenin precursor NH1 16MKANLLVLLC THWVYS 73 43 54 Neuraminidase 26 MLPSTVQTLT LLLTSGGVLL SLYVSA83 48

Adjusting the Selected MHC Alleles Repertories to the EligibleTargets/Indications

Since the evaluated antigens of the invention emerge from pathogens thatare more frequent in certain geographic territories, the MHC predictioncalculations were adjusted with dominant Class I and II alleles fromthese territories. The outcome is MHC-predictions coverage for two mainterritories: Sub Sahara Africa and South West Asia (in addition to theWestern population) for the same list of selected targets.

Tables 2 and 3 provide unique allele coverage for TB and malaria targetssuitable for the population in South-West Asia (Table 2) and Sub SaharanAfrica (Table 3).

TABLE 2 % % MHC- MHC- Protein Disease length I II  1 BPBP1 Tuberculosis26 78 66  2 Antigen 85B Tuberculosis 40 77 62  3 Antigen 85B-Precursorleprae 38 78 49  4 Lipoprotein lpqH Tuberculosis 24 77 51  5 LipoproteinLpqH 19 KDa leprae 20 60 56  6 Beta gluconase putative Tuberculosis 3181 57  7 Hypothetical protein MT0 213 Tuberculosis 27 80 58  8 ProteaseTuberculosis 34 84 55  9 ATP dependet helicase putative Tuberculosis 3281 47 10 Hypotethical protein MT1221 Tuberculosis 37 76 52 11 BCGTuberculosis 23 71 66 12 Un char protein Tuberculosis 19 85 62Rv0476/MTO4941 prec 13 Un char protein Tuberculosis 20 82 66Rv1334/MT1376 prec 14 Putative lipoprotein lprB prec Tuberculosis 25 6762 15 Putative lipoprotein lpqV prec Tuberculosis 25 78 62 16Beta-Lactomase precursor Tuberculosis 30 74 57 17 Circumsporozoitprotein P. Falciparum 18 62 66 precursor 18 Malaria exported protein-1P. Falciparum 22 85 66 19 Liver stage antigen (LSA-1) P. Falciparum 2384 66 20 Sporozoit surface antigen 2 P. Falciparum 25 80 66 21 MSP1(merozoit surface P. Falciparum 20 84 62 protein 1 precur). 22 ProteinAntigen P. Falciparum 25 80 57 23 Cytoadherence linked asexual P. Vivax24 78 66 protein, CLAG 24 Membrane protein PF12 P. Vivax 23 78 54 25Exported protein 2, putative P. Vivax 21 83 60 26Circumsporozoite-protein P. Vivax 21 77 50 related antigen, 27Circumsporozoite protein P. Vivax 22 76 66 precursor 28 Merozoitesurface protein P. Vivax 23 79 57 3 alfa 29 CTRP adhesive protein P.Vivax 23 78 66 invasiv stage

TABLE 3 % % MHC- MHC- I II  1 BPBP1 Tuberculosis 26 80 55  2 Antigen 85BTuberculosis 40 83 62  3 Antigen 85B-Precursor leprae 38 85 51  4Lipoprotein lpqH Tuberculosis 24 86 55  5 Lipoprotein LpqH 19 KDa leprae20 83 62  6 Beta gluconase putative Tuberculosis 31 89 56  7Hypothetical protein MT0 213 Tuberculosis 27 86 62  8 ProteaseTuberculosis 34 81 52  9 ATP dependet helicase putative Tuberculosis 3288 30 10 Hypotethical protein MT1221 Tuberculosis 37 89 56 11 BCGTuberculosis 23 74 62 12 Un char protein Tuberculosis 19 90 62Rv0476/MTO4941 prec 13 Un char protein Tuberculosis 20 90 62Rv1334/MT1376 prec 14 Putative lipoprotein lprB prec Tuberculosis 25 8462 15 Putative lipoprotein lpqV prec Tuberculosis 25 91 62 16Beta-Lactomase precursor Tuberculosis 30 84 56 17 Circumsporozoitprotein P. Falciparum 18 89 62 precursor 18 Malaria exported protein-1P. Falciparum 22 92 62 19 Liver stage antigen (LSA-1) P. Falciparum 2393 62 20 Sporozoit surface antigen 2 P. Falciparum 25 92 62 21 MSP1(merozoit surface P. Falciparum 20 90 62 protein 1 precur). 22 ProteinAntigen P. Falciparum 25 94 56 23 Cytoadherence linked asexual P. Vivax24 91 62 protein, CLAG 24 Membrane protein PF12 P. Vivax 23 83 57 25Exported protein 2, putative P. Vivax 21 91 62 26Circumsporozoite-protein P. Vivax 21 82 49 related antigen, 27Circumsporozoite protein P. Vivax 22 81 55 precursor 28 Merozoitesurface protein P. Vivax 23 89 56 3 alfa 29 CTRP adhesive protein P.Vivax 23 91 62 invasiv stageDetermining the Percentage of Homology Between a Selected Antigen with aSpecific Signal Peptide and Other Self Proteins

The selected putative SP Antigens were then evaluated for homology withhuman proteins using Genbank http://www.ncbi.nlm.nih.gov:80/blast/.Though most of the selected SP antigens did not show identity with humansequences, any peptide which shared greater than 80% identity withpeptides contained in the human genome would have been considered forelimination prior to selection for synthesis. Practically, similaritywas searched in the minimal class I 9mer epitopes.

Preferred antigens in accordance with the invention are listed in Tables1-3, in particular:

-   -   0. TB derived antigens, “Putative lipoprotein lpqV precursor”        (VXL214) “BPBP1” (VXL200), Hypothetical protein MTO 213        (VXL206), Protease (VXL207), having an SP of 25, 26, 27 and 34        AA long respectively, high SP score, over 50% binding for both        class I and II and no homology with human sequences. Additional        preferred TB antigens are provided in FIG. 1. Specifically those        antigens denoted as ATP dependent Helicase putative (VXL208),        Uncharacterized protein Rv0476/MT04941 precursor (VXL211),        Uncharacterized protein Rv1334/MT1376 precursor (VXL212),        Antigen 85B (VXL201) and “Lipoprotein lpqH (VXL203).    -   1. The Malaria antigens “exported protein-1”, “Sporozoit surface        antigen 2” and “MSP1” which are less than 25 AA long, have a        high SP score, over 50% for both class I and II and no homology        with human sequences.    -   2. The Toxoplasma “GRA-1” antigen.    -   3. The HCV antigen “Unique short US7 glycoprotein precursor”    -   4. The HIV “Transforming membrane receptor-like protein” antigen    -   5. Influenza Hemagglutinin precursor and Neuraminidase

Evaluation of the Selected Candidates In Vitro Analysis

Signal peptide vaccine candidates (VCs) that were selected according tothe above criteria were synthesized and partially purified (>70% purity)for initial evaluation. The evaluation process included in vitroexperiments for determination of immune properties as follows:peripheral blood mononuclear cells (PBMCs) were obtained from a largepool of healthy (PPD positive and Negative) and infected donors, e.g.individuals with active Mtb, and stimulated with the selectedmulti-epitope VCs. The stimulated PCMCs were subjected to proliferationassays evaluating three main parameters; absolute stimulation index(SI), percentage of positive SI>2 stimulations and key cytokinesecretion, primarily IFN-Gamma, IL-2, IL-4 and IL-12 as measured in anELISA assay.

Peptide VCs which tested positive in the above assays were furtherpurified (>90% purity) and subjected to further development. Additionalexperiments included comparing the activity of the pure SP-derived VCsto that of other antigen-matched epitopes derived from other domains.Separately, T cell lines directed against the most potent immunodominantmulti-epitope VCs, in accordance with the invention, were establishedfrom naïve and infected donors, and the T cell phenotype and functionwere examined.

For phenotype characterization, these T cell lines were evaluated inFACS analysis for the following cell surface markers: CD3, CD4, CD8,CD45RO, CD44, and CD62L.

For functional characterization, these T cell lines were furtherevaluated in several cytotoxicity assays. In these studies, target cellswere either autologous human macrophages loaded with one of themulti-epitope VCs or autologous macrophages infected with a livepathogen (e.g. Mtb).

Functional properties of the same T cell lines were also manifested inFACS analysis measuring in an Intra-Cellular staining (ICS) assayIFN-Gamma and IL-4 levels in CD4, and CD8 cells. This assay specificallydetermines the phenotype of the vaccine-reacting T cells subpopulations.IFN-Gamma, IL-2, IL-4 and IL-12 cytokine secretion levels were alsomeasured in the same T cell lines using ELISA assay either during theprocess of generating the T cell line or during the lysis process toconfirm a correlation between strong lysis and high IFN-Gamma secretionof the effector cells.

Constructing a Multi-Antigenic and Multi-Epitopes Recombinant ProteinContaining the Most Potent VCs

mTbuVax is an artificial recombinant protein vaccine containing the fivemost immunodominant multi-epitope VCs VXL201, VXL203, VXL208, VXL211 andVXL212 separated by flexible linkers. In addition, 5-7 differentrestriction sites were also designed in the construct to enables simpleextension of the linker and replacement of the VCs along the gene.Briefly, the designed gene was synthetically synthesized, introducedinto a plasmid, propagated and sequenced in order to verify the requiredsequence. The amino acid sequence of the recombinant protein is asfollows (SEQ ID NO: 55):

TMGSGGSGASGGSGKKKKKKKKMLLRKGTVYVLVIRADLVNAMVAHAKKKGGSGASGGSGGASGASGGSGKKKKKKKKKMLVLLVAVLVTAVYAFVHAKKKGGSGASGGSGGGSGASGGSGGGSGSGGGKKKKKKKKKMRFAQPSALSRFSALTRDWFTSTFAAPTAAQAKKKGGSGASGGSGGGSGASGVDTSGSGGSGGGSGGKKKKKKKKKMKRGLTVAVAGAAILVAGLSGCSSKKKGGSGGSGGGGSGASGGSGGGSGASGGSGGGSGASGGSGKKKKKKKKKMTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGALE

The nucleic acid sequence of the recombinant protein is as follows (SEQID NO: 56):

CCATGGgtagtggcggatctggcgcgagtggcgggagtgggaaaaagaaaaaaagaaaaagaaaATGCTGTTACGTAAAGGCACCGTTTATGTGCTGGTCATTCGCGCCGATCTTGTGAATGCGATGGTTGCACATGCGaaaaagaaaggtggcagtggtgcctctggaggttctgggggtGCTAGCggcgcgagtggtgggagcgggaagaaaaaaaagaaaaagaaaaagaaaATGCTTGTGCTGCTCGTTGCGGTTCTGGTGACCGCCGTCTACGCATTTGTCCATGCGaaaaagaaaggtggctcaggagccagtggtggaagcgggggtggctccggggcgagcggtggatctggcggagggtctggcagcggcggtgggaaaaagaagaaaaaaaagaaaaagaaaATGCGTTTCGCACAGCCGAGCGCGCTGTCTCGCTTTAGTGCACTGACCCGTGATTGGTTTACGAGCACCTTCGCCGCGCCGACTGCGGCACAGGCTaagaaaaaaggcggttctggggcgtcaggcgggagcggcggaggatcaggtgcctctggtGTCGACACTAGTggcagtggagggtctggtggaggctctggtggcaaaaaaaagaaaaagaaaaagaaaaagATGAAACGTGGCCTGACCGTTGCGGTGGCAGGTGCGGCCATTCTGGTGGCAGGTCTGAGCGGCTGCTCTAGTaagaaaaaaggagggagcggcggttcgggcggtggagggagcggtgcctctggcggttcaggtggcggaagtggggcatccggcggttccggcggtggaagcggtgcctctggaggcagtggtaagaaaaagaaaaagaaaaaaaagaaaATGACCGATGTTAGCCGCAAAATCCGTGCCTGGGGCCGTCGCCTGATGATCGGCACCGCAGCTGCGGTTGTGCTGCCGGGTCTGGTTGGCCTTGCAGGTGGCGCCGCGACCGCAGGCGCGCTCGAG

For expression and optimization, the gene in pET plasmid was introducedinto E. coli BL21 and expression of the mTbuVax protein was optimizedunder different growing conditions. Analysis includes SDS-PAGE, affinitypurification according to the tag in use and specific anti-VXL seraobtained from TB patients.

In Vivo Evaluation of Immunodominant and Protection Properties

Immunodominant properties of selected vaccine candidates are verified invivo (in particular CTL development, proliferation) in suitable micestrains, e.g. BALB/C mice, following ex vivo cytotoxicity assessments.

Additional characteristics can be evaluated in protective experiments inMtb infected mice rescued by adoptive transfer of PBLs activated ex vivowith one or more of the vaccines candidate; or in protective experimentsin Mtb infected mice immunized with vaccines candidates.

Furthermore, toxicology studies can be performed in appropriate speciesfor the selected candidate vaccines.

Terms and Definitions

The nomenclature used to describe peptide and/or polynucleotidecompounds of the invention follows the conventional practice wherein theamino group (N-terminus) and/or the 5′ are presented to the left and thecarboxyl group (C-terminus) and/or 3′ is presented to the right.

As used herein, the term “peptide vaccine” refers to a preparationcomposed of at least one peptide that improves immunity to a particularpathogen.

As used herein, the term “peptide” refers to a molecular chain of aminoacids, which, if required, can be modified in vivo or in vitro, forexample by manosylation, glycosylation, amidation (specificallyC-terminal amides), carboxylation or phosphorylation with thestipulation that these modifications must preserve the biologicalactivity of the original molecule. In addition, peptides can be part ofa chimeric protein.

Functional derivatives of the peptides are also included in the presentinvention. Functional derivatives are meant to include peptides whichdiffer in one or more amino acids in the overall sequence, which havedeletions, substitutions, inversions or additions. Amino acidsubstitutions which can be expected not to essentially alter biologicaland immunological activities have been described. Amino acidreplacements between related amino acids or replacements which haveoccurred frequently in evolution include, inter alia Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val.

The peptides according to the invention can be produced synthetically orby recombinant DNA technology. Methods for producing synthetic peptidesare well known in the art.

The organic chemical methods for peptide synthesis are considered toinclude the coupling of the required amino acids by means of acondensation reaction, either in homogenous phase or with the aid of aso-called solid phase. The condensation reaction can be carried out asfollows:

Condensation of a compound (amino acid, peptide) with a free carboxylgroup and protected other reactive groups with a compound (amino acid,peptide) with a free amino group and protected other reactive groups, inthe presence of a condensation agent;

Condensation of a compound (amino acid, peptide) with an activatedcarboxyl group and free or protected other reaction groups with acompound (amino acid, peptide) with a free amino group and free orprotected other reactive groups.

Activation of the carboxyl group can take place, inter alia, byconverting the carboxyl group to an acid halide, azide, anhydride,imidazolide or an activated ester, such as the N-hydroxy-succinimide,N-hydroxy-benzotriazole or p-nitrophenyl ester.

In one specific embodiment an amide group is added at the 3′ end of thesynthetic peptides of the invention.

The most common methods for the above condensation reactions are: thecarbodiimide method, the azide method, the mixed anhydride method andthe method using activated esters, such as described in The Peptides,Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.)1979, 1980, 1981 (Academic Press, Inc.).

Production of peptides by recombinant DNA techniques is a general methodwell known in the art. The polypeptide to be expressed is coded for by anucleic acid sequence.

Also part of the invention is the nucleic acid sequence comprising thesequence encoding the peptides according to the present invention.

As is well known in the art, the degeneracy of the genetic code permitssubstitution of bases in a codon to result in another codon still codingfor the same amino acid, e.g., the codon for the amino acid glutamicacid is both GAT and GAA. Consequently, it is clear that for theexpression of a polypeptide with an amino acid sequence as shown in anyof SEQ ID NO: 1-9 use can be made of a derivate nucleic acid sequencewith such an alternative codon composition thereby different nucleicacid sequences can be used.

The term “Nucleotide sequence” as used herein refers to a polymeric formof nucleotides of any length, both to ribonucleic acid (RNA) sequencesand to deoxyribonucleic acid (DNA) sequences. In principle, this termrefers to the primary structure of the molecule. Thus, this termincludes double and single stranded DNA, as well as double and singlestranded RNA, and modifications thereof.

The nucleotide sequences encoding the peptide vaccines of the inventioncan be used for the production of the peptides using recombinant DNAtechniques. For this, the nucleotide sequence must be comprised in acloning vehicle which can be used to transform or transfect a suitablehost cell.

A wide variety of host cell and cloning vehicle combinations may beusefully employed in cloning the nucleic acid sequence. For example,useful cloning vehicles may include chromosomal, non-chromosomal andsynthetic DNA sequences such as various known bacterial plasmids, andwider host range plasmids such as pBR 322, the various pUC, pGEM andpBluescript plasmids, bacteriophages, e.g. lambda-gt-Wes, Charon 28 andthe M13 derived phages and vectors derived from combinations of plasmidsand phage or virus DNA, such as SV40, adenovirus or polyoma virus DNA.

Useful hosts may include bacterial hosts, yeasts and other fungi, plantor animal hosts, such as Chinese Hamster Ovary (CHO) cells, melanomacells, dendritic cells, monkey cells and other hosts.

Vehicles for use in expression of the peptides may further comprisecontrol sequences operably linked to the nucleic acid sequence codingfor the peptide. Such control sequences generally comprise a promotersequence and sequences which regulate and/or enhance expression levels.Furthermore, an origin of replication and/or a dominant selection markerare often present in such vehicles. Of course, control and othersequences can vary depending on the host cell selected.

Techniques for transforming or transfecting host cells are well known inthe art (for instance, Maniatis et al., 1982/1989, Molecular cloning: Alaboratory Manual, Cold Spring Harbor Lab.).

The present invention also provides a polynucleotide encoding the signalpeptide vaccine of the invention as part of a pharmaceutical compositionfor targeted treatment of an intracellular pathogen.

Further aspects of the present invention are directed to a method fortreating or for preventing a pathogen infection by administering thepharmaceutical compositions of the present invention to a patient inneed thereof.

The peptide vaccine of the invention is administered in animmunogenically effective amount with or without a co-stimulatorymolecule. According to the method of the invention, the peptide vaccinemay be administrated to a subject in need of such treatment for a timeand under condition sufficient to prevent, and/or ameliorate thepathogen infection.

The peptide of the invention may be used in conjunction with aco-stimulatory molecule. Both molecules may be formulated separately oras a “chimeric vaccine” formulation, with a pharmaceutically acceptablecarrier and administered in an amount sufficient to elicit a Tlymphocyte-mediated immune response.

According to the methods of the invention, the peptide may beadministered to subjects by a variety of administration modes, includingby intradermal, intramuscular, subcutaneous, intravenous, intra-atrial,intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal,intrapulmonary, and transdermal delivery, or topically to the eyes,ears, skin or mucous membranes. Alternatively, the antigen may beadministered ex-vivo by direct exposure to cells, tissues or organsoriginating from a subject (Autologous) or other subject (Allogeneic),optionally in a biologically suitable, liquid or solid carrier.

In certain embodiments of the invention, the peptides or pharmaceuticalcompositions with or without a co-stimulatory molecule are delivered toa common or adjacent target site in the subject, for example to aspecific target tissue or cell population in which the vaccineformulation is intended to elicit an immune response. Typically, whenthe peptide or pharmaceutical composition and the optionalco-stimulatory molecule are administered separately, they are deliveredto the same or closely proximate site(s), for example to a single targettissue or to adjacent sites that are structurally or fluidly connectedwith one another (e.g., to allow direct exposure of the same cells,e.g., fluid flow transfer, dissipation or diffusion through a fluid orextracellular matrix of both vaccine agents). Thus, a shared target sitefor delivery of antigen and co-stimulatory molecule can be a commonsurface (e.g., a mucosal, basal or lunenal surface) of a particulartarget tissue or cell population, or an extracellular space, lumen,cavity, or structure that borders, surrounds or infiltrates the targettissue or cell population.

For prophylactic and treatment purposes, the peptide vaccine with orwithout a co-stimulatory molecule may be administered to the subjectseparately or together, in a single bolus delivery, via continuousdelivery (e.g., continuous intravenous or transdermal delivery) over anextended time period, or in a repeated administration protocol (e.g., onan hourly, daily or weekly basis). The various dosages and deliveryprotocols contemplated for administration of peptide and co-stimulatorymolecule, in simultaneous or sequential combination, are immunogenicallyeffective to prevent, inhibit the occurrence or alleviate one or moresymptoms of infection in the subject. An “immunogenically effectiveamount” of the peptide thus refers to an amount that is, in combination,effective, at dosages and for periods of time necessary, to elicit aspecific T lymphocyte mediated immune response. This response can bedetermined by conventional assays for T-cell activation, including butnot limited to assays to detect proliferation, specific cytokineactivation and/or cytolytic activity, as demonstrated in the Examplesbelow.

For prophylactic and therapeutic use, peptide antigens might beformulated with a “pharmaceutical acceptable carrier”. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption enhancing or delaying agents, and otherexcipients or additives that are physiologically compatible. In specificembodiments, the carrier is suitable for intranasal, intravenous,intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosalor transdermal administration. Depending on the route of administration,the active compound may be coated in a material to protect the compoundfrom the action of acids and other natural conditions which mayinactivate the compound.

Peptide or polypeptide vaccines may be administered to the subject perse or in combination with an appropriate auxiliary agent or adjuvant viainjection. Alternatively, the peptide or polypeptide vaccine may bepercutaneously administered through mucous membrane by, for instance,spraying the solution. The unit dose of the peptide typically rangesfrom about 0.01 mg to 100 mg, more typically between about 100micrograms to about 5 mg, which may be administered, one time orrepeatedly, to a patient.

Examples of auxiliary agents or adjuvants which can be formulated withor conjugated to peptide or protein antigens and/or vectors forexpressing co-stimulatory molecules to enhance their immunogenicity foruse within the invention include cytokines (e.g. GM-CSF), bacterial cellcomponents such as BCG bacterial cell components, imnunostimulatingcomplex (ISCOM), extracted from the tree bark called QuillA, QS-21, asaponin-type auxiliary agent, Montanide ISA 51VG, liposomes, aluminumhydroxide (alum), bovine serum albumin (BSA), tetanus toxoid (TT),keyhole limpet hemocyanin (KLH), and TLR (Toll-like receptor)-basedadjuvants (e.g. see Heit et al Eur. J. Immunol. (2007) 37:2063-2074).

In preparing pharmaceutical compositions of the present invention, itmay be desirable to modify the peptide antigen, or to combine orconjugate the peptide with other agents, to alter pharmacokinetics andbiodistribution. A number of methods for altering pharmacokinetics andbiodistribution are known to persons of ordinary skill in the art.Examples of such methods include protection of the proteins, proteincomplexes and polynucleotides in vesicles composed of other proteins,lipids (for example, liposomes), carbohydrates, or synthetic polymers.For example, the vaccine agents of the invention can be incorporatedinto liposomes in order to enhance pharmacokinetics and biodistributioncharacteristics. A variety of methods are available for preparingliposomes, as described in, e.g., U.S. Pat. Nos. 4,235,871, 4,501,728and 4,837,028. For use with liposome delivery vehicles, peptides aretypically entrapped within the liposome, or lipid vesicle, or are boundto the outside of the vesicle.

Within certain embodiments of the invention, peptide antigens areassociated with liposomes, such as lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture, or the DNA may beassociated with an adjuvant known in the art to boost immune responses,such as a protein or other carrier. Additional agents which assist inthe cellular uptake of DNA, such as, but not limited to, calcium ions,viral proteins and other transfection facilitating agents and methodsmay also be used to advantage.

Examples Measurement of Proliferation Responses Induced by VXLTuberculosis Vaccine Candidates (VC)

Peripheral blood mononuclear cells (PBMC) from 11 naïve human donorswere individually stimulated with 10 g/ml of partially purified (>70%),VXL tuberculosis VCs for 6 days. On day 6, 0.5 μCi/well of ³[H]Thymidine was added for an additional incubation of 18 hours. Sampleswere harvested and the radioactive counts were measured in a β-counter.The list of VXL VCs is presented in FIG. 1. The controls used in thisstudy were phytohaemagglutinin (PHA), tuberculosis purified proteinderivative (PPD) and tetanus toxoid (TT) for positive stimulation andHu-Fab fragment as a negative stimulation (Morgan E L and Weigle W O(1981) J Exp Med: 154778-790). Results presented in FIG. 2 are indicatedas positive stimulation index SI≥2 (A) or as an average SI (B) from anaverage of 8 different experiments using 11 naïve donors. SI wascalculated by dividing the CPM obtained in an analyzed sample to the CPMin control medium sample. Results revealed strong SI of 2.6+/−1 to4+/−3.3 for VXL201, VXL203, VXL208, VXL211 and VXL212 (FIG. 2B) withpositive (SI≥2) proliferation to these peptides in 54%-81% of thedonors. The results were statistically significant as measured in aT-Test analysis (p<0.05 or lower) for all the vaccine candidates tested,except for VXL200 (FIG. 2A) as compared to the Hu-Fab negative control.Conclusion: VCs VXL 201, 208, 211, 212 and to some extent also 203showed the highest SI and widest coverage in the total 11 donorsevaluated, in comparison with the positive controls. The results supportthe pan MHC coverage of the VXL VCs of the invention.

FIG. 3 represents an experiment in which the proliferative properties ofthe VXL tuberculosis vaccine candidates shown in FIG. 2 were furtheranalyzed only on Tetanus Toxoid (TT) or Tuberculosis Purified proteinderivative (PPD) positive responded (SI>2) naïve PBMC. Namely, on PBMCobtained from donors previously exposed to the tuberculosis bacteria orvaccinated against the tetanus antigen or tuberculosis. The experimentwas performed as described above. Conclusion: The VCs VXL 200, 201, 208,211, 212 are stronger in healthy donors which also manifested responseto PPD i.e. they are potentially more associated with anti-tuberculosisimmunity. In parallel, VCs VXL 201, 208, 211, 212 and to some extentalso 214, 200 and 203 are more positive in donors, which also manifestedresponse (SI>2) to TT i.e. they are active in an immunocompetent system.

FIG. 4 represents an experiment in which the proliferative properties ofthe VXL tuberculosis vaccine candidates shown in FIG. 2 was furtheranalyzed only on naive PBMC derived from PPD positive versus PPDnegative donors. The experiment was performed as described above.Conclusion: VCs VXL 201, 208, 211, 212 are stronger antigens in naïvedonors and are even stronger in donors with previous exposure to thetuberculosis Bacteria. VXL 203 is more potent in donors which were notexposed in the past to the tuberculosis Bacteria. There is a role forboth types of antigens in the development of preventive vs. therapeuticvaccine.

We next conducted similar proliferation studies with peripheral bloodmononuclear cells (PBMC) from 14 patients with active Mtb. Thecharacterization of these tuberculosis patients and in particular theirethnic group and disease-associated parameters are described in FIG. 5.Similar to other proliferation studies, controls used in this study werePHA, PPD and TT for positive stimulation and Hu-Fab as a negativestimulation. Proliferation results presented in FIG. 6 are indicated aspositive stimulation index SI≥2 (A) or as an average SI (B) from anaverage of 4 different experiments using 14 TB patients. Resultsrevealed extremely strong SI of 3.2+/−3.4 to 8.0+/−6.8 for VXL201,VXL203, VXL211 and VXL212 with positive (SI>2) in 54-100% of thepatients evaluated. The proliferation in TB patients was significantlystronger compared to that in naïve donors suggesting a primaryrecognition/priming against the VXL VCs by the patient's immune system.The results were statistically significant (T-Test p<0.05 or lower) forall the vaccine candidates (excluding VXL208) tested as compared to theHu-Fab negative control. In addition results are also significant (Ttest p<0.05 or lower) for the vaccines candidate VXL201, VXL203, VXL211,VXL212 as compared to the other vaccine candidates.

Measurement of Cytokine Secretion Responses Induced by VXL TuberculosisVaccine Candidates (VCs)

In order to define the Th1 or Th2 profile during the stimulation processwith the different VXL VC, PBMC derived from 11 naïve donors wereindividually stimulated once by all the partially purified (>70% pure)tuberculosis VCs for 48 hrs and supernatant was used to evaluatecytokine levels in a quantitative ELISA assay. Results in FIG. 7Apresent the percentage of donors with positive (x>0.1 ng/ml) cytokinesecretion from the 11 evaluated naïve donors. FIG. 7B presents one ofthree similar experiments for cytokine secretion and proliferationresponse to VXL203, VXL208, VXL211, VXL212 using 5 of the 11 donors.Results in FIG. 7A shows that IL-2 secretion is observed followingstimulation with each of the VXL VCs but widely ranged from 25% of thedonors with VXL200 to 91.6% with VXL211. IL-4 secretion was much lowerand observed only following stimulation with VC VXL 207 and 208 in 25%of the donors. IL-12 secretion levels ranged from 8.6% with VXL203, 206,207 to 58.3% in VXL 211 and VXL212. IFN-gamma and TNF alfa secretionlevels ranged from 8.6-50% and 8.6-41.6% with the same VCs. Theseresults indicate that the best VCs for inducing Th1 immunity in thesedonors were VXL211 and VXL212 and to some extent also VXL 201, VXL203and VXL208. In the second experiment, FIG. 7B, the cytokine secretionanalysis showed a broad secretion (1.5-3.3 ng/ml) of IL-2, IL-12,TNF-alfa, IFN-gamma and to a lesser extent IL-4 by the VXL203, VXL208,VXL211, VXL212 VCs. In these donors, the high secretion levels werecorrelated with strong SI.

FIG. 8A, B presents similar experiments conducted with PBMCs derivedfrom 6 tuberculosis patients. Results in FIG. 8A present a lowpercentage of positive (x>0.1 ng/ml) cytokine secretion from the 6evaluated donors with active TB. Results in FIG. 8B revealed lower(1.12-1.89 ng/ml) secretion which was limited mainly to TNF-alfa andIFN-gamma by VCs VXL211 and VXL212. In addition, unlike in naïve donorsin these patients there was no correlation between the low levels andthe strong SI values. Conclusion: In naïve donors, VXL203, VXL208,VXL211, VXL212 and to some extent VXL201 were the most potent VCs ininducing high secretion of IL-2, IL-12 and/or IFN-gamma which are keyTh1 cytokines associated with anti-TB immunity. Among these VCs onlyVXL208 induced some secretion of IL-4 which can lead to a mix Th1 andTh2 immunity combining T cells and antibodies. In these studies,specific cytokine secretion was mostly correlated with high SI inproliferation analysis in the majority of the evaluated donors. On thecontrary, high SI scores in patients with active tuberculosis wereusually associated with lower secretion levels of mainly IL-12 andIFN-gamma and were observed only in few of the donors. As already shownby (Torres M et al., 1998) this phenomenon represents the poor immunestatus of these patients and inhibition of immunological function ofperipheral lymphocytes by the M. Tuberculosis (Dietrich J. et al 2009)and set a higher bar for any future anti-TB therapeutic vaccine.

Based on the above described initial proliferation and cytokinesecretion experiments, the VCs VXL 201, 203, 208, 211 and 212 wereselected for further evaluation. These multi-epitope peptides werere-synthesized, further purified (purity>90%) and revalidated in 2proliferation experiments for bioactivity/specificity in comparison tothe same semi-purified (>70% pure) peptides. Experiments were conductedas previously described on six additional healthy naïve donors and 4 TBpatients. The results shown in FIG. 9 present higher SI scores for thepure VCs in naïve donors (FIG. 9A) and TB patients (FIG. 9A). Among thedifferent VCs a significant elevation in proliferation potency wasmanifested mainly by VXL203 with average SI levels of 1.8+/−0.8 in thepartially purified peptide compared with 5.7+/−6.8 for pure peptide onnaïve donors. Moderate elevation was also observed in proliferationstudies performed in PBMC derived from TB patients PBMC FIG. 9B.Conclusion: Results confirmed the potency and specificity of theselected immunodominant VCs in naïve individuals and in TB patients.

To further confirm the superior immunogenic properties of the SignalPeptide derived multi-epitope peptides VXL201, VXL203, VXL208, VXL211,VXL212, their proliferation properties were compared with controlpeptides derived from other domains in the same antigens. FIG. 10represents one of two similar experiments in which the proliferativeeffect of VXL tuberculosis VCs was compared to that of other known ornovel epitopes derived from the same tuberculosis antigens. PBMCs from 6naïve donors were individually stimulated for 6 days by crude VXL201(>70% purity) and pure (>90% purity) VXL203, VXL208, VXL211, VXL212 oralternative known and novel pure (>90% purity) peptide epitopes derivedfrom the same antigens. The alternative non SP-derived peptide epitopeswere selected and scored according to their predicted MHC binding asfollows: A-high, B-medium, or C-Low MHC class I binding. The length ofpreviously published alternative epitopes was kept as is; while thelength of novel alternative epitopes was adjusted to that of theevaluated antigen matched VXL VCs. Results suggested that proliferationis significantly higher with all VXL VC vs. their alternativeantigen-match control VCs. While VXL VCs manifested absolute average SIscore ranging from 4-7, each alternative VCs excluding VXL201Amanifested negative SI≤2. These results are highly significant (T-Testp<0.01 or lower) for any VXL VC vs. its alternative antigen-match VCexcluding VXL201 vs. 201A (P=0.077). One potential explanation for theresults obtained with VXL201 VC could be related to its final purity(70% vs. 90% for other peptides). Comparable results were achieved in asimilar analysis conducted with TB patients (data not shown).Conclusions: The SP-derived multi-epitope VCs VXL 201, VXL203, VXL208,VXL211, VXL212 are far more immunodominant than any other known or novelepitope selected from a different domain on the same tuberculosisantigen. Moreover, these results show that the ability of inducingimmunity in heterogeneous population of naïve or TB patients-derivedPBMC is mostly restricted to SP-derived VCs.

Next, an in depth analysis of the phenotype and function of T cell linesgenerated against the five selected VCs was performed. T-cell lines weregenerated against the five individual VXL VCs as follows: 3 naïve donorsagainst VXL203, 2 naïve donors against VXL208, 3 naïve donors VXL211, 2naïve donors against VXL212 and one patient against VXL211. T cellgeneration was performed by initial stimulation of PBLs with VC-pulsedautologous DC, followed by stimulation with autologous peptide-loadedmacrophages and a final stimulation with soluble VC. The establishedT-cell lines were then evaluated for their cytotoxic activity.Autologous Macrophages were loaded for 18 hrs with 50 g/ml VXL VCs,Control antigen match VCs, or PPD, labeled for 14 hours with 35SMethionine and used as targets. An additional control used wasnon-loaded Macrophages. FIG. 11 presents an average cytotoxic activityin three different experiments using 2 or 3 different donors.Cytotoxicity at 50:1 E:T ratio by VXL203, 208, 211 and 212-induced Tcells against the same evaluated VXL VC (peptide) was very strong andhighly specific at the range of 90-100% lysis vs. 5-30% of the differentcontrols including the alternative antigen-match non-SP peptides andunloaded targets. T cell lines induced against VXL203 and VXL211 alsomanifested specific lysis against PPD-loaded DC suggesting the presenceof VXL203 and VXL211 in the PPD's antigenic repertoire. Results arehighly significant (T-Test p<0.01 or lower) for each VXL VC vs. itsalternative antigen-match epitopes A or B and PPD excluding VXL203 andVXL211 vs. PPD.

Next, the lysis properties of the same peptide-induced T cell linesagainst autologous macrophages (Mϕ) infected with live Mtb wasevaluated. M were infected for 18 h with live bacteria at a ratio of1:10 M: bacteria in RPMI complete Medium without antibiotics. Next, themedium was removed and the M were further incubated for additional 48 hin RPMI complete medium with the G418 antibiotic which specificallyinhibits the extracellular growth of the Mtb bacteria. For thecytotoxicity assay infected M (target cells) were washed and cultivatedat a ratio of 1:100 and/or 1:50 with VXL-induced T cell lines(effectors) for 5 h. At the final step, the bacteria growth medium wascollected, separated from cells, washed, diluted 1:1000 in RPMI mediumand 1 ml was used to seed the bacteria on dedicated dishes withMiddlebrool 7H9 solid medium. For evaluating the spontaneous lysis,infected M were incubated with complete RPMI medium containing an equalnumber of non stimulated PBMCs from donors or patients. For total lysis,infected m were treated with 1 ml of 10% Tryton X100. Dishes wereincubated in 37° C. for 2 weeks and colony forming units (CFU) werecounted. The percentage of specific lysis was calculated as follows: No.of colonies in Exp. dish (-) No. of colonies in spontaneous dish (/) No.of colonies in Total dish (-) No. of colonies in spontaneous dish.

FIGS. 12A and B summarize the cytotoxicity properties of the naïve andpatient-derived T-cell lines. The results suggest high and specificlysis by anti-VXL201 and anti-VXL203 and moderate lysis by anti VXL211 Tcell lines in both naïve and TB patients. Anti-VXL208 and VXL212 T cellline manifested lower lysis levels in these studies. FIG. 13A, Bsummarizes the positive VCs-specific donor and patient T cells as wellas their range of activity in the cytotoxicity assay. As an additionalparameter for potency, levels of the Th1 cytokine IFN-gamma released bythe anti-VXL201, 203, 211 effector T-cells lines during the lysisprocess, were measured in an ELISA assay. Very significant levels ofIFN-gamma secretion by both naïve and TB-patient-derived T cell lines inparticular anti-VXL201 and anti-VXL203 were detected, as follows: Fordonor #14 (FIG. 12A): upon stimulation with VXL201, 6 μg/ml IFN-gammawere detected; upon stimulation with VXL203, 16 μg/ml IFN-gamma weredetected and upon stimulation with VXL211, 9.2 μg/ml IFN-gamma weredetected.

For donor #15 (FIG. 12B): upon stimulation with VXL203, 3.8 μg/mlIFN-gamma were detected; upon stimulation with VXL208, 3.6 μg/mlIFN-gamma were detected and upon stimulation with VXL211, 5.9 μg/mlIFN-gamma were detected.

For patient #17 (FIG. 12C): upon stimulation with VXL201, 10 μg/mlIFN-gamma were detected; upon stimulation with VXL203, 12 μg/mlIFN-gamma were detected, upon stimulation with VXL208, 2.6 μg/mlIFN-gamma were detected, upon stimulation with VXL211, 7.8 μg/mlIFN-gamma were detected, and upon stimulation with VXL212, 5.6 μg/mlIFN-gamma were detected.

For patient #18 (FIG. 12D): upon stimulation with VXL201, 14 μg/mlIFN-gamma were detected; upon stimulation with VXL203, 11 μg/mlIFN-gamma were detected, and upon stimulation with VXL212, 7.5 μg/mlIFN-gamma were detected.

For patient #19 (FIG. 12E): upon stimulation with VXL201, 11.3 μg/mlIFN-gamma were detected; upon stimulation with VXL203, 4.6 μg/mlIFN-gamma were detected, and upon stimulation with VXL212, 3.6 μg/mlIFN-gamma were detected.

This intensive secretion was correlated with the high specific lysis ofthese T cell lines. Conclusions: The five selected VXL VCs in particularVXL 201, 203, 211 are potent inducers of IFN-gamma secretion from T celllines with specific and robust lysis capability of both peptide-loadedand bacteria infected targets. Moreover, cytotoxicity results confirmedthe processing and presentation of the VXL peptides on the surface oftarget cells. More importantly, the effective lysis of T cell linesisolated from tuberculosis patient suggest that the VXL VCs are potentcandidates as therapeutic vaccines in addition to serving as apreventive vaccine, i.e. transforming non-responsive PBMC with limitedcytokine secretion properties to functional T cells with high lysis andcytokine secretion properties. Last but not least, these results alsovalidate the ability of Mtb antigenic epitopes to be efficientlypresented on the host cells in association with MHC molecules, andthereby allow their specific destruction.

The phenotype of the T cell population during development and at thefinal stage whereby they are used in cytotoxicity studies, wereevaluated by FACS analysis.

FIG. 14, presents results of the evaluation of four anti-VXL VCs T celllines developed from 2 naïve donors. The CD4+ or CD8+ positive T cellpopulation expressing the effector cell marker CD44^(high) and effectormemory marker CD62L^(high) was examined.

Results shown in FIG. 14 demonstrate a significant increase inCD8+/CD44^(high) and CD8+/CD62L^(high) subpopulation after the 3^(rd)stimulation with each evaluated anti-VXL VCs T-cell line in particularVXL203 and VXL211. The increase in CD44^(high) activated effector cellsranged from 79% to 80% and from 17% to 40.9% in CD62L^(high) memorycells for VXL203 and VXL211 respectively. VXL208 manifested a lowerincrease in CD44^(high) activated effector cells 50.2% and 32.1% inCD62L^(high). CD4+ T cells demonstrated an increase in CD4+/CD44^(high)and CD4+/CD62L^(high) subpopulation already after the 1^(st)stimulation. This level wasn't augmented following the 2^(nd) and 3^(rd)stimulations. The levels of CD44^(high) activated effector cells rangedfrom 41% for VXL11 to 52% for VXL208 and from 22% for VXL208 to 39% forVXL 203 in CD62L^(high) memory cells. Without wishing to be bound bytheory, an explanation for these results can be related to the initialpriming of CD4+ cells mediated by the Mtb.

Next, the percentage of IFN-gamma and IL-4 producing CD4+ and CD8+T-cells was measured in ICS. For this FACS study the maturedanti-VXL201, 203 and 211 T cell lines after the 3^(rd) stimulation,developed from two naïve donors, were used. T cell lines were stimulatedfor 6 h with autologous m loaded with the corresponding VXL peptide orantigen-match peptide as a negative control. For achieving a maximalcytokine production the T cells lines were stimulated with 50 ng/ml PMAand 750 ng/ml lonomycine.

Results presented in FIG. 15, show high and specific IFN-gamma producingT cells following stimulation with VXL Peptide and none followingstimulation with the antigen match control peptides. The maximalpercentage of specific IFN-gamma produced by CD8+ T-cells was inanti-VXL211 (10.6%) FIG. 15C and anti-VXL203 (8.2%) FIG. 15A upper leftas compared to a lower production by 203A FIG. 15B and 211A FIG. 15D.IFN-gamma production was manifested by both CD4+ and CD8+ subpopulation.The same T cell line didn't manifest any IL-4 secretion followingstimulation with VXL Peptide and the antigen match control peptides(data not presented). Conclusions: VXL VCs 201, 203, 208 and 211 induceda robust and antigen-specific immune activation by both T-cellssubpopulation CD4+ and CD8+. This response was mediated by CD44^(high)activated effector cells and CD62L^(high) memory cells suggesting T celldifferentiation to memory post activation. Further more, these cellsdemonstrated high functionally for INF-gamma (Th1) but not IL-4 (Th2)production in ICS, lysis of peptide-loaded and MTB infected autolugues mand IFN-gamma secretion (during the lysis).

1. A method for producing a peptide vaccine against a pathogen, themethod comprising: synthesizing a peptide not longer than 40 amino acidscomprising a sequence of a signal peptide (SP) of a selected antigenicprotein, wherein said selected antigenic protein is selected due to itbeing expressed by a host cell infected by said pathogen, and comprisinga SP, thereby producing a peptide vaccine against a pathogen.
 2. Themethod of claim 1, wherein said pathogen is an intracellular pathogen.3. The method of claim 2, wherein said intracellular pathogen is abacterium or a virus.
 4. The method of claim 2, wherein said pathogen isselected from tuberculosis, malaria, toxoplasma, human immunodeficiencyvirus (HIV) and hepatitis C virus and influenza.
 5. The method of claim1, wherein said antigenic protein is further selected due to at leastone of: having increased expression in pathogenic cells as compared tonon-pathogenic cells; being antigen protein eligible for an immuneassault; and being an antigen protein with an SP sequence with predictedbinding to an HLA class I or class II protein.
 6. The method of claim 5,wherein a protein eligible for an immune assault is: a proteintransported through the ER; a non-ubiquitously expressed protein; anon-immune related protein; and a protein expressed in a cell thatcomprises MHC presentation.
 7. The method of claim 5, wherein said HLAclass I or class II protein is an HLA class I or class II protein highlyexpressed by a population infected by said pathogen.
 8. The method ofclaim 1, further comprising prior to said synthesizing ruling out a SPsequence comprising greater than 80% identity with a human peptide thatis not the antigenic protein target.
 9. The method of claim 8, wherein aminimal class I 9mer epitope from said SP comprising greater than 80%identity is ruled out.
 10. The method of claim 1, wherein said peptidenot longer than 40 amino acids comprises a minimal class I 9mer epitopeof said SP.
 11. The method of claim 1, wherein said selected antigenicprotein is further selected due to its stimulating proliferation ofperipheral blood mononuclear cells (PBMCs) in vitro.
 12. The method ofclaim 11, wherein stimulating proliferation comprises at least one of:increasing absolute stimulate index (SI); increasing the percentage ofpositive SI greater than 2 stimulations; and increasing cytokinesecretion.
 13. The method of claim 10, wherein said cytokine is selectedfrom: Interferon Gamma, Interleukin-2 (IL-2), IL-4, and IL-12.
 14. Themethod of claim 11, wherein said PBMCs are from a healthy donor or adonor infected with said pathogen.
 15. The method of claim 1, furthercomprising purifying said synthesized peptide.
 16. The method of claim1, further comprising synthesizing a multi-antigenic peptide comprisinga sequence of an SP of a first selected antigenic proteins and asequence of an SP of a second selected antigenic protein, wherein saidfirst and said second antigenic proteins are both expressed in a cellinfected with said pathogen and wherein each sequence comprising a SPsequence is not greater than 40 amino acids.
 17. The method of claim156, wherein said multi-antigenic peptide comprises a linker betweensaid SP of a first selected antigenic protein and said SP of a secondselected antigenic protein.
 18. The method of claim 1, wherein saidsynthesizing comprises culturing a host cell comprising one or morevectors comprising a nucleic acid sequence encoding said peptide.
 19. Avaccine produced by the method of claim
 1. 20. A method of producing anenriched T cell population activated against a pathogen, comprisingadministering to an enriched T cell population the vaccine of claim 19.